A quantitative molecular model for modulation of mammalian translation by the eIF4E-binding protein 1

Karim, M.M., Hughes, J.M.X., Warwicker, J., Scheper, G.C., Proud, C.G. and McCarthy, J.E.G. (2001) A quantitative molecular model for modulation of mammalian translation by the eIF4E-binding protein 1. The Journal of Biological Chemistry, 276 (23), 20750-20757. (doi:10.1074/jbc.M011068200).

Abstract

Translation initiation is a key point of regulation in eukaryotic gene expression. 4E-binding proteins (4E-BPs) inhibit initiation by blocking the association of eIF4E with eIF4G, two integral components of the mRNA cap-binding complex. Phosphorylation of 4E-BP1 reduces its ability to bind to eIF4E and thereby to compete with eIF4G. A novel combination of biophysical and biochemical tools was used to measure the impact of phosphorylation and acidic side chain substitution at each potentially modulatory site in 4E-BP1. For each individual site, we have analyzed the effects of modification on eIF4E binding using affinity chromatography and surface plasmon resonance analysis, and on the regulatory function of the 4E-BP1 protein using a yeast in vivo model system and a mammalian in vitro translation assay. We find that modifications at the two sites immediately flanking the eIF4E-binding domain, Thr46 and Ser65, consistently have the most significant effects, and that phosphorylation of Ser65 causes the greatest reduction in binding affinity. These results establish a quantitative framework that should contribute to understanding of the molecular interactions underlying 4E-BP1-mediated translational regulation.

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